FOR IN VITRO DIAGNOSTIC USE

Transcription

1 For the qualitative detection and identification of the influenza A, influenza B, respirary syncytial virus (RSV), adenovirus, parainfluenza 1, parainfluenza 2 and parainfluenza 3 virus in respirary specimens. FOR IN VITRO DIAGNOSTIC USE INTENDED USE The Diagnostic Hybrids, Inc. D 3 Ultra DFA (direct fluorescent antibody) Respirary Virus Screening & ID Kit is intended for the qualitative detection and identification of the influenza A, influenza B, respirary syncytial virus (RSV), adenovirus, parainfluenza 1, parainfluenza 2 and parainfluenza 3 virus in respirary specimens, by either direct detection or cell culture method, by immunofluorescence using fluoresceinated monoclonal antibodies (MAbs). It is recommended that specimens found be negative after examination of the direct specimen result be confirmed by cell culture. Negative results do not preclude respirary virus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions. Performance characteristics for influenza A were established when influenza A/H3 and A/H1 were the predominant influenza A viruses in circulation. When other influenza A viruses are emerging, performance characteristics may vary. If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent state or local health departments for testing. Viral culture should not be attempted in these cases unless a BSL3+ facility is available receive and culture specimens. 1 SUMMARY AND EXPLANATION OF THE TEST With the addition of new antiviral drugs for the treatment of influenza 2, more rapid and sensitive tests for respirary virus detection, 3,4 and the increasing need be more discriminating in the use of antibiotics 5, early detection and identification of the infecting viral agent has grown substantially in importance. Viral identification is becoming increasingly important in ruling out bacteria as the cause of respirary infections. Virus identification by either direct antigen detection or cell culture using fluorescent monoclonal antibodies continues be the standard method in virology laboraries. Influenza Virus Influenza viruses (family Orthomyxoviridae) contain a single-stranded RNA genome which is present in 8 separate segments of ribonucleoprotein. This segmentation of the genome is rare among viruses and probably D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 1 of 30

2 contributes the rapid development of new influenza strains through interchange of gene segments if two different viruses infect the same cell. There are 3 types of influenza, A, B and C. Type A has counterparts in birds and pigs as well as humans, while types B and C are known only in man. Due the possibility of another pandemic caused by influenza A, as occurred in 1918 when million people worldwide died, the Centers for Disease Control (CDC) and the World Health Organization (WHO) maintain surveillance of influenza strains and make predictions of suitable strains for vaccine production. Influenza infects an estimated 120 million people in the US, Europe and Japan each year and it is estimated that in the US there are 75,000 deaths annually from pneumonia caused by influenza. Primary viral pneumonia or pneumonia from secondary bacterial infections are the primary causes of morbidity of the viral infection. 6 Pandemics of influenza A occur about every years and epidemics of either influenza A or B occur annually. Infections are seasonal, typically extending from November April in the northern hemisphere. Complications tend occur in the young, elderly and persons with chronic cardio-pulmonary diseases. Incubation time is 1-3 days with rapid spread by inhalation via aerial droplets and fomites. It is characterized by fever, myalgia, headache and pharyngitis. Influenza A and B are most commonly isolated in A549/Mv1Lu mixtures (R-Mix ), A549/MDCK mixtures (R-Mix Too ), Rhesus MK, MDCK, MRC-5 and A549 cells. Adenovirus Adenoviruses (family Adenoviridae) are non-enveloped, double stranded DNA viruses. There are 49 serotypes, further divided in 6 groups, A F, with most associated with respirary and ocular infections. Generally, adenovirus infections in adults have a low morbidity with the exceptions of immunocompromised patients and individuals living in cramped quarters where infections can cause atypical pneumonia. Virus spread is commonly via aerial droplets and fomites where they infect the mucous membranes of the eye, respirary tract and gut. 7 Adenovirus can be isolated in A549/Mv1Lu mixtures (R-Mix), A549/MDCK mixtures (R-Mix Too), HEp2, HEK, A549 and MRC-5 cells. 7 Parainfluenza Virus Parainfluenza viruses (family Paramyxoviridae) are enveloped viruses with a single, negative strand RNA genome. The 4 different types, 1 4, cause croup and viral pneumonia in children under the age of 5 years and cause upper respirary illness in adults. Parainfluenza is the number 2 leading cause of lower respirary illness in children (after RSV). Outbreaks caused by parainfluenza viruses occur during alternate years in the fall (P1 and P2) or throughout the year, with increased activity in the spring (P3). 8 Parainfluenza viruses can be isolated in A549/Mv1Lu mixtures (R-Mix), A549/MDCK mixtures (R-Mix Too), Rhesus MK, MRC-5 and LLC-MK2 cells. Trypsin is helpful in the medium for recovery of types 1 and 2 but not type 3. 7 Respirary Syncytial Virus (RSV) RSV (family Paramyxoviridae) is an enveloped virus with a single, negative strand RNA genome. RSV infections cause viral bronchiolitis and pneumonia in infants and the common cold in adults. 9 RSV is usually a seasonal (winter and early spring) infection with epidemics lasting up 5 months. Peak mortality due RSV occurs in 3-4 month old infants. There are two major subtypes, A and B; Subtype B is characterized as the asympmatic strain that the majority of the population experiences. The more severe clinical illnesses involve Subtype A D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 2 of 30

3 strains which tend predominate in most outbreaks. 10 RSV is the primary viral cause of lower respirary disease in infants and young children. Re-infections do occur but tend be limited minor upper respirary infections. 11 RSV is also now recognized as a significant problem in certain adult populations. These include the elderly, persons with cardiopulmonary diseases, and immunocompromised hosts. 12 RSV is commonly detected directly in cells from the nasopharyngeal epithelium by staining with immunofluorescent reagents 10 although it can be isolated in cell cultures of A549/Mv1Lu mixtures (R-Mix), A549/MDCK mixtures (R-Mix Too), HEp2, Vero, LLC-MK2 and MRC-5 cells. 7 PRINCIPLE OF THE PROCEDURE The D 3 Ultra DFA Respirary Virus Screening & ID Kit uses viral antigen-specific murine monoclonal antibodies that are directly labeled with fluorescein for the rapid detection and identification of respirary viruses. The kit includes a DFA Screening Reagent that contains a blend of murine monoclonal antibodies (MAbs) directed against seven respirary viruses (influenza A, influenza B, respirary syncytial virus, adenovirus, parainfluenza 1, parainfluenza 2, and parainfluenza 3) plus seven separate DFA Reagents, each consisting of MAb blends directed against a single respirary virus. The kit can be used for direct specimen or cell culture screening and final virus identification. The cells be tested, either derived from a clinical specimen or cell culture, are fixed in acene. The DFA Screening Reagent is added the cells determine the presence of viral antigens. After incubating at 35 C 37 C, the stained cells are rinsed with the diluted Wash Solution (1X). A drop of the supplied Mounting Fluid is added and a coverslip is placed on the prepared cells. The cells are examined using a fluorescence microscope. Virus infected cells will be stained with viral specific apple-green fluorescence when stained with the DFA Screening Reagent while non-infected cells will contain no fluorescence but will be stained red by the Evans Blue counter-stain. If the specimen contains fluorescent cells, the particular virus is identified using the separate DFA Reagents on new, separate cell preparations. If on examination of a direct stained specimen, no fluorescent-stained cells are found and all the cells stain red from the Evans Blue, it is recommended that the specimen be cultured and stained using the DFA Screening Reagent. If fluorescent cells are seen, the identification of the virus is determined as described above. Cell preparations are fixed in acene. The individual DFA Reagents are added the cell preparations. After incubating at 35 C 37 C, the stained cells are rinsed with the diluted Wash Solution (1X). A drop of the supplied Mounting Fluid is added and a coverslip is placed on the stained cells. The cells are examined using a fluorescence microscope for the presence of viral specific apple-green fluorescence. The unknown respirary virus is then identified and reported. REAGENTS AND MATERIALS PROVIDED The D 3 Ultra DFA Respirary Virus Screening & ID Kit contains the following: Respirary Virus Screening Reagent 10 ml One dropper bottle containing a blend of fluorescein labeled murine monoclonal antibodies directed against respirary viral antigens of influenza A, influenza B, respirary syncytial virus (RSV), adenovirus, parainfluenza 1, parainfluenza 2 and parainfluenza 3. The buffered, stabilized, aqueous solution contains Evans Blue as a counter-stain and 0.1% sodium azide as preservative. Influenza A DFA Reagent 2 ml One dropper bottle containing fluorescein labeled murine monoclonal antibodies directed against antigens produced by influenza A virus (Texas 1/77, H3N2 strain) infected cells. The buffered, stabilized, aqueous solution contains Evans Blue as a counter-stain and 0.1% sodium azide as preservative. D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 3 of 30

4 Influenza B DFA Reagent 2 ml One dropper bottle containing fluorescein labeled murine monoclonal antibodies directed against antigens produced by influenza B virus (Hong Kong 5/72 strain) infected cells. The buffered, stabilized, aqueous solution contains Evans Blue as a counter-stain and 0.1% sodium azide as preservative. RSV DFA Reagent 2 ml One dropper bottle containing fluorescein labeled murine monoclonal antibodies directed against antigens produced by RSV (Long strain) infected cells. The buffered, stabilized, aqueous solution contains Evans Blue as a counter-stain and 0.1% sodium azide as preservative. Adenovirus DFA Reagent 2 ml One dropper bottle containing fluorescein labeled murine monoclonal antibodies directed against antigens produced by adenovirus (Type 3-GB strain and Type 6-nsil 99 strain) infected cells. The buffered, stabilized, aqueous solution contains Evans Blue as a counter-stain and 0.1% sodium azide as preservative. Parainfluenza 1 DFA Reagent 2 ml One dropper bottle containing fluorescein labeled murine monoclonal antibodies directed against antigens produced by parainfluenza virus type 1 (VP-1 strain) infected cells. The buffered, stabilized, aqueous solution contains Evans Blue as a counter-stain and 0.1% sodium azide as preservative. Parainfluenza 2 DFA Reagent 2 ml One dropper bottle containing fluorescein labeled murine monoclonal antibodies directed against antigens produced by parainfluenza virus type 2 (Greer strain) infected cells. The buffered, stabilized, aqueous solution contains Evans Blue as a counter-stain and 0.1% sodium azide as preservative. Parainfluenza 3 DFA Reagent 2 ml One dropper bottle containing fluorescein labeled murine monoclonal antibodies directed against antigens produced by parainfluenza 3 (C243 strain) infected cells. The buffered, stabilized, aqueous solution contains Evans Blue as a counter-stain and 0.1% sodium azide as preservative. Respirary Virus Antigen Control Slides 5 slides Five individually packaged control slides containing wells with cell culture derived positive and negative control cells. Each positive well is identified as the virus infected cells present, i.e., influenza A, influenza B, respirary syncytial virus (RSV), adenovirus, parainfluenza 1, parainfluenza 2 and parainfluenza 3. The negative well contains non-infected cells. Each slide is intended be stained only one time. Normal Mouse Gamma Globulin DFA Reagent 10 ml One dropper bottle containing a mixture of fluorescein labeled murine gamma globulin that has been shown be un-reactive with any of the listed respirary viruses. The buffered, stabilized, aqueous solution contains Evans Blue as a counter-stain and 0.1% sodium azide as preservative. 40X Wash Solution Concentrate 25 ml One bottle of 40X PBS concentrate consisting of Tween 20 and 4% sodium azide (0.1% sodium azide after dilution 1X using de-mineralized water). Mounting Fluid 15 ml One dropper bottle containing an aqueous, buffered, stabilized solution of glycerol and 0.1% sodium azide. MATERIALS REQUIRED BUT NOT PROVIDED Fluorescence microscope with the correct filter combination for FITC (excitation peak = 490 nm, emission peak = 520 nm); magnification 200X 400X Cell culture for respirary virus isolation. Suggested cell lines include LLC-MK2, HEp-2, A549 cells, R-Mix and R-Mix Too MixedCells, and primary Rhesus monkey kidney cells (all are available from Quidel) Coverslips (22 x 50 mm) for Antigen Control Slides and for specimen slides D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 4 of 30

5 Universal Transport Medium (available from Quidel) R-Mix Refeed Medium (for use with R-Mix and R-Mix Too MixedCells) or other standard refeed medium (available from Quidel) Reagent-grade acene (> 99% pure) chilled at 2 C 8 C for fixation of direct specimen slides, shell-vials and cultured cell preparations NOTES: Keep the reagent-grade acene container tightly sealed avoid hygroscopic absorption of water, which may cause a hazy, non-specific, background fluorescence. A mixture of 80% acene/20% de-mineralized water is used for fixing cells in plastic multi-well plates. Sre at ambient temperature (20 C 25 C). Sterile graduated pipettes: 10 ml, 5 ml, and 1 ml Sterile Pasteur pipettes or other transfer pipettes CAUTION: One should not use solvents such as acene with polyethylene transfer pipettes. Fine-tipped forceps Wash bottle, 200 ml Bent-tip teasing needle (for removal of coverslip from a shell-vial for the typing portion of the procedure); fashion the teasing needle by bending the tip of a syringe needle or similar object (e.g., mycology teasing needle) against a benchp or with a pair of forceps taking care avoid injury. Sodium hypochlorite solution (1:10 final dilution of household bleach) Humid chamber (e.g., covered Petri dish with a damp paper wel placed in the botm) Glass microscope slides Acene-cleaned multi-well glass microscope slides (2-well and 8-well masked slides) Blotters for multi-well glass microscope slides 2- and 8-well absorbent blotters, used blot excess liquid from the mask prevent spread of liquid or stained cells from one well the other Sterile, nylon flocked swabs or polyester swabs, which are non-inhibiry viruses and cell cultures Incubar, 35 C 37 C (5% CO 2 or non-co 2, depending on the cell culture format used) Centrifuge with free-swinging bucket ror De-mineralized water for dilution of 40X Wash Solution Concentrate and for dilution of the reagent-grade acene for use in polystyrene multi-well plates PBS, sterile, for use in rinsing and suspending cells Live control viruses for positive culture controls Known strains of the 7 respirary viruses for use in moniring the cell culture and staining procedures; such control virus strains can be obtained from Quidel. Aspirar Set-up Vacuum aspirar with disinfectant trap containing sufficient household bleach (5%) that the concentration is not decreased by more than 10-fold as it is diluted with discarded fluids Wash Container Beaker, wash bottle or Coplin jar for washing slides Fixing Container Coplin jar, slide dish or polyethylene holder for slides for use in fixing the cells on the slides Inverted Light Microscope Used for examining the monolayers of cells prior inoculation and examination for xicity, confluency and for cypathic effects (CPE); it should have between 40X 100X magnification capability. WARNINGS AND PRECAUTIONS For in vitro diagnostic use. No known test method can offer complete assurance that infectious agents are absent; therefore, all human blood derivatives, reagents and human specimens should be handled as if capable of transmitting infectious disease. It is recommended that reagents and human specimens are handled in accordance with the OSHA Standard on Bloodborne Pathogens. D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 5 of 30

6 Cell culture isolation may have some potential be hazardous. Personnel working with cell cultures must be properly trained in safe handling techniques 13,14,15 and have experience with cell culture before attempting this procedure. All procedures must be conducted in accordance with the CDC 5 th Edition Biosafety in Microbiological and Biomedical Laboraries, 2007, and CLSI Approved Guideline M29-A, Protection of Laborary Workers from Instrument Biohazards and Infectious Disease Transmitted by Blood, Body Fluids, and Tissue. All specimens and materials used process them should be considered potentially infectious and handled in a manner which prevents infection of laborary personnel. Biosafety Level 2 or other appropriate biosafety practices should be used when handling these materials. Decontamination of specimens and cultures is most effectively accomplished using a solution of sodium hypochlorite (1:10 final dilution of household bleach). Although Antigen Control Slides have been shown be non-infectious, the same precautions taken in handling and disposing of other infectious materials should be employed in their use. Never pipette reagents or clinical samples by mouth; avoid all contact of clinical samples with broken skin. Avoid splashing and the generation of aerosols with clinical samples. Use aseptic technique and sterile equipment and materials for all tissue culture procedures. Acene, a reagent that is required for the test but not provided in the kit, is a flammable, volatile organic solvent. Use it in a well-ventilated area and keep away from flames and other sources of ignition. Sodium azide is included in the 40X PBS Concentrate at a concentration of 4% (w/v), and in the other solutions in this kit at 0.1% concentration. Sodium azide is considered poisonous. If the 40X PBS Concentrate is swallowed, seek medical advice immediately. Aqueous solutions of sodium azide, when mixed with acids, may liberate xic gas. Avoid disposal of these solutions down sanitary or industrial plumbing systems. Sodium azide may react with lead and copper plumbing form highly explosive metal azides. Avoid release the environment. Evans Blue counter-stain is a potential carcinogen. If skin contact occurs, flush with water immediately. The DFA Reagents are supplied at working strength. Any dilution of the reagents will decrease sensitivity. Reagents should be used prior their expiration date. Each Respirary Virus Antigen Control Slide should be used only once. Do not re-use a control slide. Microbial contamination of the DFA Reagents may cause a decrease in sensitivity. Sre 1X Wash Solution and PBS in a clean container prevent contamination. Reusable glassware must be washed and thoroughly rinsed free of all detergents. Do not expose the DFA Reagents bright light during staining or srage. Use of reagents other than those specified with the components of this kit may lead erroneous results. Testing should be performed in an area with adequate ventilation. Dispose of containers and unused contents in accordance with Federal, State and Local regulary requirements. Wear suitable protective clothing, gloves, and eye/face protection when handling the contents of this kit. Wash hands thoroughly after handling. For additional information on hazard symbols, safety, handling and disposal of the components within this kit, please refer the Safety Data Sheet (SDS) located at quidel.com. PREPARATION OF 1X WASH SOLUTION After srage at 2 C 8 C, some salts in the 40X Wash Solution Concentrate may have crystallized. Warm the solution room temperature (20 C 25 C) re-dissolve the crystals, then mix. D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 6 of 30

7 Add contents of the fully dissolved 25 ml 40X Wash Solution Concentrate 975 ml of de-mineralized water. Label the 1X Wash Solution with a sixty (60) day expiration date after reconstitution and sre at ambient temperature. STORAGE Table 1. Reagent Srage Conditions Respirary Virus DFA Screening Reagent Influenza A DFA Reagent Influenza B DFA Reagent RSV DFA Reagent Adenovirus DFA Reagent Parainfluenza 1 DFA Reagent Parainfluenza 2 DFA Reagent Parainfluenza 3 DFA Reagent Mounting Fluid Normal Mouse Gamma Globulin DFA Reagent Respirary Virus Antigen Control Slides 40X Wash Solution Concentrate NOTE: The Concentrate may crystallize when sred at 2 C 8 C. The crystals will dissolve when the Concentrate is warmed room temperature. 1X Wash Solution Sre at 2 C 8 C in the dark. Sre at 2 C 8 C. Sre liquid at 2 C 8 C prior dilution. Sre at room temperature (20 C 25 C). STABILITY Reagents and components will retain their full potency through the expiration date shown on the label of each bottle when sred at recommended temperatures. Light exposure of the DFA Reagents should be kept a minimum. Discard 1X Wash Solution if it becomes cloudy. SPECIMEN COLLECTION AND PREPARATION Proper collection and handling of the patient specimen are the most important facrs in successful respirary virus detection. Specimen collection, specimen processing, and cell culture of viruses should be attempted only by personnel that have been trained in such procedures. Care should be taken during all specimen collection and handling avoid generation of aerosols. For specimen collection and processing recommendations, refer CLSI Approved Viral Culture Guidelines. 16 Collection 17 Aspirates and Washes containing secretions from the nasopharyngeal epithelium provide the best specimens for direct specimen testing since they will contain large numbers of epithelial cells. Aspirates can be collected using a sterile, soft polyethylene #8 infant feeding tube attached a disposable aspiration trap connected a suction device. Washes can be collected by instilling and aspirating 1 2 ml of saline in the patient s nostril while the patient is in a supine position. D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 7 of 30

8 Aspirates and washes should be diluted with equal volumes of transport medium contained in a centrifuge tube with several sterile glass beads. Swabs from nasal, throat and nasopharyngeal areas often do not contain sufficient numbers of columnar epithelial cells allow for direct specimen detection of respirary viruses. Specimen Transport and Srage All potentially infectious agents should be transported according International Air Transport Association (IATA), International Civil Aviation Organization, (ICAO), Titles 42 and 49 of the US Code of Federal Regulations, or other regulary requirements, as may be applicable. Specimens should be transported the laborary at 2 C 8 C. This temperature can be attained by using cold packs, wet ice, foam refrigerant, or other coolants. 18 The specimens should be processed and tested as soon as possible and then sred at 2 C 8 C. Specimens should be sred at 2 C 8 C for no longer than 2 days before being tested. If longer srage is required, the specimens should be frozen at 70 C or lower. Freezing and thawing of specimens should be avoided since this will result in a loss of viability of viruses, leading decreased sensitivity of the test. PROCEDURE Preliminary Comments and Precautions Adhere the recommended volumes and times in the following procedure ensure that accurate results are obtained. For specimen swabs received in transport medium with glass beads, vortex vigorously for about 15 seconds dissociate adhered cells. For swabs not received in transport medium, transfer them a tube of transfer medium containing glass beads and vortex vigorously for about 15 seconds dissociate adhered cells. When staining with fluorescent reagents and examining cells microscopically for fluorescence, it is very important include controls, both positive and negative, monir the procedure and performance of the reagents. It is recommended that such controls be run with each batch of patient specimens. Place the closed, humidified chamber for holding slides during staining in the incubar for equilibration 35 C 37 C prior staining. By doing this, the test slides and reagents will come temperature quickly, yielding more rapid, intense staining. Bring DFA Reagents ambient temperature (20 C 25 C) prior use, and immediately return refrigerar after use for srage at 2 C 8 C. Regarding Cell Culture Testing Good Laborary Practice dictates that positive and negative virus controls be run with each new batch of cells confirm their performance in culturing specific viruses. It is good practice retain the medium removed from the monolayers until after staining results have been obtained. If there is any question concerning the specimen results, the medium can be passed another monolayer and incubated for the appropriate time period for repeat testing. When using cell cultures in polystyrene multi-well plates, dilute the acene fixative 80% by adding 20 ml of de-mineralized water 80 ml of acene. (See MATERIALS REQUIRED BUT NOT PROVIDED.) Do not allow the monolayers dry before fixing; this can lead high background staining and decreased sensitivity. Do not allow the DFA Reagents dry on the monolayers; this can lead high background. Regarding Immunofluorescence Microscopy D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 8 of 30

9 Examine the positive and negative controls before examining the test specimens. If one of these fails perform as expected, review the steps and conditions under which the test was performed determine the root cause(s). Do not report results for patient samples until controls perform as expected. Three aspects of the fluorescence microscope must be functioning properly and optimally in order achieve maximum brightness of fluorescence: The activation light source has a finite life and as it ages, its output decreases, resulting in lower fluorescence intensity from the DFA Reagent. The light source is focused by a number of lenses and mirror(s). For maximum intensity, these must be properly aligned. The filters used in the light path must be appropriate for the particular fluor, in this case, fluorescein. Fluorescent artifacts may be observed in the cell monolayers: Cell debris, lint, etc. can non-specifically adsorb the DFA Reagent, resulting in highly intense fluorescence. These can be identified by their morphology, i.e., they don t have the appearance of a complete cell and typically are not seen on the same plane of the monolayer as the other cells would be. A low grade, yellow-green fluorescence may sometimes be seen, particularly in areas that have piled cells or are near holes in the cell monolayer. In both cases, the diffusion of the entrapped DFA Reagent is retarded during the wash step, resulting in the non-specific fluorescence. Intense fluorescence around the periphery of slide wells is indicative of drying of the DFA Reagent during incubation, suggesting that it was incubated o long or the humidity was not well controlled. Non-specific fluorescence caused by adsorption of the DFA Reagent trapped by inadequate removal of mucus from direct specimens. Trapping of fluorescence by leukocytes and monocytes may occur on direct specimens. Likewise, RBCs in the specimen may leave a green haze on the sample. Inadequate washing can lead general low grade fluorescence due residual DFA Reagent remaining on the monolayer of cells Protect stained slides and monolayers from light as much as possible during testing. Bleaching or fading of the fluorescence of stained cells may occur on exposure light, particularly light of high intensity. This bleaching can occur when a stained cell is viewed in a fluorescence microscope for an extended period of time. Specimen Preparation For specimen processing recommendations, refer CLSI Approved Viral Culture Guidelines Vortex the specimen vigorously for seconds. 2. Centrifuge at xg for 5 10 minutes. 3. Collect and set aside the supernatant for viral isolation. (See step #10 below.) 4. Add 5 ml of PBS and vortex vigorously for seconds. 5. Centrifuge at xg for 5 10 minutes. 6. Remove the supernatant and the mucus layer above the cell pellet taking care not disturb the cell pellet. 7. Repeat steps 4 through 6 until the mucus layer has been completely removed. NOTE: It is important remove all the mucus since it can cause non-specific fluorescence. 8. Add ml of PBS. 9. Mix the suspension by pipetting up and down re-suspend the cell pellet, forming a slightly cloudy suspension. This cell suspension will be used for Direct Specimen Testing. NOTE: The quality of the slide preparation is dependent on the concentration of cells in the suspension; o many cells make it difficult read the result and o few decrease the sensitivity of the procedure. Specimens may also be centrifuged if a monolayer is preferred. D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 9 of 30

10 10. For use in Cell Culture Testing, add the supernatant that was reserved in Step #3, the cell suspension that remains after Direct Specimen Testing. Add a few sterile glass beads the tube and vortex for about 15 seconds break up the cells and release any virus. Repeat this step for each specimen. Direct Specimen Testing 1. Spot 25 µl of the prepared cell suspension 20 on each well of a 2-well and an 8-well slide. Repeat this step for each specimen. 2. Air-dry the wells completely. 3. Fix the cells the slides using fresh, chilled acene for 5 10 minutes at 20 C 25 C. CAUTION: Acene is volatile and flammable; keep away from open flames. 4. Remove the slides from the fixative and allow air dry. 5. Add one drop of the DFA Screening Reagent completely cover the dried, fixed cells on one well of each of the 2-well slides. 6. Also, each of the wells of a fresh Respirary Virus Antigen Control Slide add one drop of the DFA Screening Reagent. An Antigen Control Slide should be stained only once, as it contains individual wells of viral infected cells and non-infected cells. 7. Add one drop of the Normal Mouse Gamma Globulin DFA Reagent completely cover the dried, fixed cells on the other well of each of the 2-well slides. 8. Place the slides in a covered humidified chamber at 35 C 37 C for minutes. 9. Rinse the stained cells using the 1X Wash Solution. For only a few slides, this can be done using a beaker of the 1X Wash Solution. For many slides, a slide carrier that holds slides can be placed in its container of 1X Wash Solution. For effective rinsing, dip the slide(s) up and down a minimum of four times. 10. Discard the used wash and repeat the washing step using new 1X Wash Solution. 11. Rinse the stained cells using de-mineralized water. For only a few slides, this can be done using a beaker of the de-mineralized water. For many slides, a slide carrier that holds slides can be placed in its container with de-mineralized water. For effective rinsing, dip the slide(s) up and down a minimum of four times. 12. Gently blot the excess de-mineralized water. 13. Add a small drop of Mounting Fluid each cell-containing well and cover the wells with a coverslip. 14. Examine the stained, mounted cells using a fluorescence microscope with magnifications between 200X 400X. (See Regarding Immunofluorescence Microscopy) 15. Refer INTERPRETATION OF RESULTS. 16. If the result is positive for respirary virus, the staining procedure may be repeated using the reserved 8- well specimen slide in order identify which respirary virus may be present. Add one drop of each individual virus DFA Reagent its corresponding well on the 8-well specimen slide. Leave one well as a negative. For the Respirary Virus Antigen Control Slide, add one drop of each individual virus DFA Reagent its corresponding labeled well. NOTE: An Antigen Control Slide should be stained only once, do not re-stain. Continue with steps 8 through 15, above. Cell Culture Testing Tube Culture 1. Examine the monolayers for proper morphology prior inoculation. 2. Aspirate maintenance medium from the monolayers and add ml of each prepared specimen (see Specimen Preparation) each of the cell lines used for respirary virus culture. 3. Place the tubes at an angle sufficient for the monolayers be covered by the inoculum; then place tubes in an incubar for 1 hour at 35 C 37 C allow virus adsorption occur. 4. After adsorption, add 2 ml of appropriate refeed medium. D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 10 of 30

11 5. Incubate the tubes at 35 C 37 C in a roller drum at 1 3 rpm. Examine the monolayers daily for evidence of xicity or viral CPE or test for hemadsorption. 6. When the monolayers are ready be stained, remove the medium by aspiration and gently rinse the monolayer two times with 1 2 ml PBS. 7. Add 0.5 ml of PBS the tube and prepare a suspension of the cells by scraping the monolayer using a pipette and breaking the cell aggregates up by pipetting the PBS up and down several times. 8. Prepare cell spots using about 25 μl of the suspension on each well of a 2-well and an 8-well slide. Repeat this step for each specimen. 9. Air dry the wells completely. 10. Fix the cells the slides using fresh, chilled acene. Let stand for 5 10 minutes, at 20 C 25 C. CAUTION: Acene is volatile and flammable; keep away from open flames. 11. Remove the slides from the fixative and allow air dry. 12. Add one drop of the DFA Screening Reagent completely cover the dried, fixed cells on one well of each of the 2-well slides. 13. Also, each of the wells of a fresh Respirary Virus Antigen Control Slide, add one drop of the DFA Screening Reagent. An Antigen Control Slide should be stained only once, as it contains individual wells of viral infected cells and non-infected cells. 14. Place the slides in a covered chamber at 35 C 37 C for minutes. 15. Rinse the stained cells using the 1X Wash Solution. For only a few slides, this can be done using a beaker of the 1X Wash Solution. For many slides, a slide carrier that holds slides can be placed in its container with 1X Wash Solution. For effective rinsing, dip the slide(s) up and down a minimum of four times. 16. Discard the used wash and repeat the washing step using new 1X Wash Solution. 17. Rinse the stained cells using de-mineralized water. For only a few slides, this can be done using a beaker of the de-mineralized water. For many slides, a slide carrier that holds slides can be placed in its container with de-mineralized water. For effective rinsing, dip the slide(s) up and down a minimum of four times. 18. Remove the de-mineralized water by aspiration. 19. Gently blot the excess liquid. 20. Add a small drop of Mounting Fluid each cell-containing well and cover the wells with a coverslip. 21. Examine the stained, mounted cells using a fluorescence microscope with magnifications between 200X 400X. (See Regarding Immunofluorescence Microscopy.) 22. Refer INTERPRETATION OF RESULTS. 23. If the result is positive for respirary virus, the staining procedure may be repeated using the reserved 8- well specimen slides in order identify which respirary virus may be present. Add one drop of each individual virus DFA Reagent its corresponding well on the 8-well specimen slide. Leave one well as a negative. For the Respirary Virus Antigen Control Slide, add one drop of each individual virus DFA Reagent its corresponding labeled well. NOTE: An Antigen Control Slide should be stained only once, do not re-stain. Continue with steps 14 through 21 above. Cell Culture Testing Shell-Vial 1. Calculate the number of shell-vials needed based on the staining procol be used (this staining procol requires 3 shell-vials): One shell-vial is required for each day the culture will be screened with the DFA Screening Reagent (i.e., staining at hours, and again at hours, requires 2 shell-vials). One additional shell-vial is required for preparing 8-well slides used identify the viruses from positive screens. 2. Examine the monolayers for proper morphology prior inoculation. D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 11 of 30

12 3. Aspirate maintenance medium from the monolayers and add 1 ml of appropriate refeed medium each shell-vial. 4. Add ml of prepared specimen each shell-vial. 5. Centrifuge the shell-vials at 700xg for 1-hour at 20 C 25 C. 6. Place sppered shell-vials in an incubar at 35 C 37 C. 7. When a monolayer is ready be stained using the DFA Screening Reagent, remove the medium by aspiration and add 1 ml of PBS. 8. Swirl mix and then aspirate. 9. Repeat this wash with another 1 ml of PBS and then aspirate. 10. Add 1 ml of fresh, chilled acene and allow stand for 5 10 minutes at 20 C 25 C. CAUTION: Acene is volatile and flammable; keep away from open flames. 11. Remove the fixative by aspiration. 12. Add 0.5 ml of PBS wet the monolayer. 13. Swirl and then aspirate completely. 14. Add 4 drops of the DFA Screening Reagent the fixed monolayers of patient and control samples, and rock ensure complete coverage of the monolayer by the Reagent. 15. Place sppered shell-vials in a 35 C 37 C incubar for minutes. 16. Aspirate the DFA Screening Reagent from the monolayers. 17. Add 1 ml of the 1X Wash Solution. 18. Remove the 1X Wash Solution by aspiration, repeat the wash step, and again remove by aspiration. 19. Add 1 ml of de-mineralized water. 20. Remove the de-mineralized water by aspiration. 21. Lift the coverslip from the botm of the shell-vial using a bent-tip needle on a syringe barrel. Grasping it with the fine-tipped forceps, transfer it, monolayer-side down, a small drop of Mounting Fluid on a standard microscope slide. 22. Examine the stained monolayers using a fluorescence microscope with magnifications between 200X 400X. (See Regarding Immunofluorescence Microscopy.) 23. Refer INTERPRETATION OF RESULTS. 24. If the result is positive for respirary virus, process a reserved replicate culture as a cell suspension and spot on an 8-well specimen slide in order identify which respirary virus may be present (See Cell Culture Testing Tube Culture steps 6 through 11, for procedure prepare a specimen slide), then: Add one drop of each individual virus DFA Reagent its corresponding well on the 8-well specimen slide. Leave one well as a negative. For the Respirary Virus Antigen Control Slide, add one drop of each individual virus DFA Reagent its corresponding labeled well. NOTE: An Antigen Control Slide should be stained only once, do not re-stain. Continue with steps 14 through 21 from Cell Culture Testing Tube Culture procedure. Cell Culture Testing Multi-Well Plate 1. Calculate the number of wells needed for the staining procol be used (this staining procol requires 3-wells): 2. One well is required for each day the culture will be screened with the DFA Screening Reagent (i.e., staining at hours, and again at hours, requires 2-wells). 3. One additional well is required for preparing 8-well slides used identify the viruses from positive screens. 4. It is recommended that each replicate well be on a different multi-well plate. This allows each plate be processed on the appropriate day. 5. Examine the monolayers for proper morphology prior inoculation. 6. Aspirate maintenance medium from the monolayers and add 1 ml of appropriate re-feed medium each 24-well multi-well plate monolayer; add 0.8 ml each 48-well plate monolayer. D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 12 of 30

13 7. Add ml of prepared specimen the appropriate wells of a multi-well plate. 8. Centrifuge the multi-well plates at 700xg for 1 hour at 20 C 25 C. 9. Place the covered multi-well plates in a 35 C 37 C incubar with a humidified, 5% CO 2 atmosphere. 10. When a monolayer is ready be stained using the DFA Screening Reagent, remove the medium by aspiration and add 1 ml of PBS. 11. Swirl mix and then aspirate. 12. Repeat this wash with another 1 ml of PBS and then aspirate. 13. Add 1 ml of 80% aqueous acene and let stand 5 10 minutes at 20 C 25 C. NOTE: Do not allow the 80% acene fixative remain in the polystyrene wells longer than 10 minutes since it may craze and cloud the plastic, making it difficult examine the monolayers. 14. CAUTION: Acene is volatile and flammable; keep away from open flames. 15. Remove the fixative by aspiration. 16. Add 0.5 ml of the PBS wet the monolayer. 17. Swirl and then aspirate completely. 18. Add 4 drops of the DFA Screening Reagent the fixed monolayers of patient and control samples in each well of a 24-well plate; add 3 drops of the DFA Screening Reagent the fixed monolayers of patient and control samples in each well of a 48-well plate. Rock ensure complete coverage of the monolayer by the Reagent. 19. Place the covered multi-well plate in a 35 C 37 C, humidified incubar for minutes. 20. Aspirate the DFA Screening Reagent from the monolayers. 21. Add 1 ml of the 1X Wash Solution and mix wash. 22. Remove the 1X Wash Solution by aspiration, repeat the wash step, and again remove by aspiration. 23. Add 1 ml of de-mineralized water. 24. Remove the de-mineralized water by aspiration. 25. Add 2 3 drops of Mounting Fluid each monolayer, then cover the plate. 26. Examine the stained monolayers using a fluorescence microscope with magnifications between 200X 400X. (See Regarding Immunofluorescence Microscopy.) 27. Refer INTERPRETATION OF RESULTS. 28. If the result is positive for respirary virus, process a reserved replicate culture as a cell suspension and spot on an 8-well specimen slide in order identify which respirary virus may be present (See Cell Culture Testing Tube Culture steps 6 through 11, for procedure prepare a specimen slide), then: Add one drop of each individual virus DFA Reagent its corresponding well on the 8-well specimen slide. Leave one well as a negative. For the Respirary Virus Antigen Control Slide, add one drop of each individual virus DFA Reagent its corresponding labeled well. NOTE: An Antigen Control Slide should be stained only once, do not re-stain. Continue with steps 14 through 21 from Cell Culture Testing Tube Culture procedure. Quality Control Reagents A fresh Respirary Virus Antigen Control Slide should be stained each time the staining procedure is performed ensure proper test performance. The positive wells will show multiple infected cells of bright apple-green fluorescence with negative cells staining a dull red due the included Evans Blue counter-stain. The negative well will show only negative cells staining a dull red. Positive and negative controls must demonstrate appropriate fluorescence for specimen results be considered valid. Antigen Control Slides may also aid in the interpretation of patient specimens. D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 13 of 30

14 The Normal Mouse Gamma Globulin DFA Reagent is used rule out those rare instances where patient cells are present that non-specifically bind the Fc portion of the mouse gamma globulin in direct specimens, which could lead a false positive result. Cell Culture Positive and negative virus controls should be run with each new batch of cells confirm their performance in culturing specific viruses. To ensure viral sensitivity, virus-inoculated control monolayers should be included each time a new lot of cell culture is used. A non-inoculated monolayer from each lot should be stained serve as a negative control. Adverse srage conditions or handling procedures will also be reflected in the negative control. If control cultures fail perform correctly, results are considered invalid. INTERPRETATION OF RESULTS Examination of Samples and Controls Examine controls first ensure proper test performance before examining patient specimens. A positive reaction is one in which bright apple-green fluorescence is observed in the infected cells. Non-infected cells will fluoresce dull red due the Evans Blue counter-stain included in the DFA Reagent. Examine the entire cell spot or monolayer of cells before reporting final results. Do not report results for patient samples unless controls perform as expected. Artifacts of Staining Dried edges of the monolayer or cell clumps may non-specifically fluoresce due antibody trapping. Dead, rounded cells may non-specifically fluoresce dull olive-green due specimen xicity or improper cell srage. Properly controlled humidity during staining and adequate washing between steps helps prevent nonspecific staining. Fluorescent Staining Pattern of Respirary Virus Infected Cells The following describes typical fluorescent patterns and should be used as a guide identify specific viruses. Note that specific viral identification requires the demonstration of characteristic staining with MAbs. The typical apple-green fluorescence staining pattern for each virus is as follows: Influenza A and B Virus The fluorescence is cyplasmic, nuclear or both. Cyplasmic staining is often punctate with large inclusions while nuclear staining is uniformly bright. Respirary Syncytial Virus The fluorescence is cyplasmic and punctate with small inclusions in the syncytia. Adenovirus The fluorescence is cyplasmic and punctate or bright nuclear or both. Parainfluenza virus types 1, 2, and 3 The fluorescence is cyplasmic and punctate with irregular inclusions. Types 2 and 3 cause the formation of syncytia. Co-infection with more than one infecting virus present in the specimen has been reported in a number of studies. The presence of multiple viruses is indicated when more than one well of the 8-well slide has fluorescent cells. The identification of the viruses is based on the individual virus DFA Reagents showing fluorescence. In such a case, it should be reported as and detected by direct specimen testing. or and isolated by cell culture. D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 14 of 30

15 Results of Direct Specimen Staining Evaluation of sample suitability Each stained patient specimen should be reviewed for the presence of columnar epithelial cells (cells that are taller than they are wide). The quality of the specimen with respect the number of epithelial cells in the sample can be assessed by examining different fields at a magnification of 200X. A satisfacry specimen should have at least 2 columnar epithelial cells per field. A negative result is indicated by the absence of fluorescence in a minimum sampling of 20 columnar epithelial cells. An inadequate specimen is indicated by fewer than 20 columnar epithelial cells present in the sample, in which case the test is considered invalid. A new specimen should be obtained and tested or cell culture for virus isolation should be initiated from the remaining specimen. Reporting Results of Direct Specimen Staining The entire cell spot must be examined for virus-infected, apple-green fluorescent cells. A satisfacry specimen with no fluorescent cells observed should be reported as No influenza A, influenza B, respirary syncytial virus, adenovirus, parainfluenza type 1, parainfluenza type 2, or parainfluenza type 3 viral antigens detected by direct specimen testing. However, such negative results should be confirmed using cell culture. Specimens negative by direct specimen testing but yielding positive culture results should be reported as isolated by cell culture, where is the appropriate virus, e.g., influenza A, influenza B, adenovirus, respirary syncytial virus, parainfluenza virus type 1, 2, or 3 (see Results from Culture Isolation/Confirmation). If apple-green fluorescent cells are found, the identification of the virus(es) may be based on the individual DFA Reagents (see PROCEDURE Direct Specimen Testing). The individual virus DFA Reagent that yields fluorescent cells represents the identification of the respirary virus. In such a case, it should be reported as detected by direct specimen testing, where is the appropriate virus, e.g., influenza A, influenza B, adenovirus, respirary syncytial virus, parainfluenza virus type 1, 2, or 3. Results from Culture Isolation/Confirmation The entire cell spot or monolayer of cells must be examined for virus-infected, apple-green fluorescent cells. If no fluorescent cells are found, the results should be reported as, No influenza A, influenza B, respirary syncytial virus, adenovirus, parainfluenza virus type 1, parainfluenza virus type 2, or parainfluenza virus type 3 isolated in cell culture. If apple-green fluorescing cells are found, the identification of the virus(es) may be based on the individual DFA Reagents (see the appropriate PROCEDURE sections). In such cases, identification of the viral antigen(s) should be reported as isolated in cell culture, where is the appropriate virus, e.g., influenza A virus, influenza B virus, respirary syncytial virus, adenovirus, parainfluenza virus type 1, parainfluenza virus type 2, or parainfluenza virus type 3. LIMITATIONS OF THE PROCEDURE Inappropriate specimen collection, srage, and transport may lead false negative culture results. 21 Assay performance characteristics have not been established for direct specimen staining on specimens other than respirary specimens. It is the user s responsibility establish assay performance for specimens other than respirary specimens. Incubation times or temperatures other than those cited in the test instructions may give erroneous results. Detection of viruses will vary greatly depending upon the specimen quality and subsequent handling. A negative result does not exclude the possibility of virus infection. Results of the test should be interpreted D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 15 of 30

16 in conjunction with information available from epidemiological studies, clinical evaluation of the patient and other diagnostic procedures. The effects of antiviral therapy on the performance of this kit have not been established. The MAbs used in this kit are from hybridomas created using viral infected cells as the immunogen. The specific viral antigens detected by the antibodies are undetermined. Since the MAbs have been prepared using defined virus strains, they may not detect all antigenic variants or new strains of the viruses, should they arise. MAbs may fail detect strains of viruses which have undergone minor amino acid changes in the target epipe region. The MAbs used in this kit are not group-specific and therefore cannot be used differentiate among the different types of adenovirus and RSV. The viral antigens detected in some direct specimens may be from non-viable virus and cannot be isolated by culture. This is particularly true of RSV which is known for its instability and loss of viability. A negative direct specimen should be inoculated in an appropriate cell culture and incubated isolate and identify any respirary virus that may be present in the specimen. A negative result on a direct or cultured specimen does not rule out the presence of virus. Performance of the kit can only be assured when components used in the assay are those supplied by Quidel. Prolonged srage of the DFA Reagents under bright light will decrease the staining intensity. Light background staining may occur with specimens contaminated with Staphylococcus aureus strains containing large amounts of protein A. Protein A will bind the F c portions of conjugated antibodies. Such binding can be distinguished from viral antigen binding on the basis of morphology, i.e., S. aureus-bound fluorescence appears as small (~1 micron diameter), bright dots. Results from cell cultures with bacterial contamination must, therefore, be interpreted with caution. EXPECTED VALUES Respirary virus infections are often seasonal, with influenza typically extending from November April in the northern hemisphere, and adenovirus infections occurring more often during late winter early summer. RSV is usually a seasonal (winter and early spring) infection as well, with epidemics lasting up five months, while outbreaks caused by parainfluenza viruses may occur throughout a year. The clinical studies described in SPECIFIC PERFORMANCE CHARACTERISTICS were comprised of respirary specimens collected during the winter early spring months of 2005/2006. Prevalence of the respirary viruses within the population of specimens that were prospectively collected and tested fresh is noted in Table 2 below (also, see Study 1-DS in SPECIFIC PERFORMANCE CHARACTERISTICS). Table 2. Prevalence of the Respirary Viruses Within the Study Population Expected Values Adeno Flu A Flu B Para 1 Para 2 Para 3 RSV Fresh Specimens (n=326) Prevalance 5.5% 9.8% 5.8% 0.6% 0% 1.5% 5.5% SPECIFIC PERFORMANCE CHARACTERISTICS This study included eight hundred and forty nine (849) original specimens evaluated by this product ( Subject device) and a currently-marketed DFA Screening & ID Kit ( Comparar device). All 849 specimens were studied by Direct Specimen (DS) testing with 22 of these specimens having insufficient cell numbers be evaluated, and one other which could not be evaluated because it exhibited non-specific staining from the Normal Mouse Gamma Globulin DFA Reagent; 520 of the specimens also were studied by Cell Culture (CC) method with one specimen not evaluated because it produced a xic cell culture monolayer. All but 30 of the D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 16 of 30

17 specimens were prospectively collected during the season; those 30 specimens had been archived as parainfluenza-positive. In addition, a set of 81 clinical isolates were tested by CC methods only. The evaluations were conducted at three laborary sites: (1) A reference laborary in northeast United States; (2) A hospital laborary in northeast United States; and (3) An internal reference laborary using specimens collected from an external hospital laborary. Percent Agreement between the Subject and Comparar devices was calculated for prospectively collected specimens. For the Respirary Virus DFA Screening Reagent: By DS method using fresh specimens, positive percent agreement is 95.5% and negative percent agreement is 98.3% (see Table 3). By DS method using frozen specimens, both positive percent agreement and negative percent agreement are (see Table 4). By CC method using frozen specimens, both positive percent agreement and negative percent agreement are (see Table 5). [See individual study results, below] Table 3. Overall Direct Specimen Results (Fresh Specimens) DS Fresh 326 Specimens Negative Screen + Adeno Flu A Flu B Para 1 Para 2 Para 3 RSV Comparar Results Subject Results Positive Percent Agreement* (PPA) 95% CI** PPA Negative Percent Agreement** (NPA) 95.5% 89.0% 98.2% 82.4% 89.3% 82.4% 34.2% 34.2% 82.4% 98.3% 98.7% 96.7% 95% CI NPA 95.7% 99.3% 95.2% 94.2% 92.9% 99.8% 96.0% 96.1% 90.8% 98.9% 95.2% * Positive Percent Agreement, or PPA, values were calculated according {[(Total Number of Positive Results in Agreement by both Subject and Comparar Devices) divided by [(Total Number of Positive Results in Agreement by both Subject and Comparar Devices) plus (Number of Results Positive by Comparar but Negative by Subject)]} multiplied by. ** Negative Percent Agreement, or NPA, values were calculated according {[(Total Number of Negative Results in Agreement by both Subject and Comparar Devices) divided by [(Total Number of Negative Results in Agreement by both Subject and Comparar Devices) plus (Number of Results Negative by Comparar but Positive by Subject)]} multiplied by. D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 17 of 30

18 Table 4. Overall Direct Specimen Results (Frozen Specimens) DS Frozen 474 specimens Negative Screen + Adeno Flu A Flu B Para 1 Para 2 Para 3 RSV Comparar Results Subject Results PPA 95% CI PPA 97.8% 63.1% 95.7% 82.3% 38.3% 38.3% 70.1% 93.0% NPA 95% CI NPA 98.8% 97.7% 95.6% 97.6% 97.8% Table 5. Overall Cell Culture Results (Frozen Specimens) 97.8% 97.6% 96.7% CC Frozen 490 specimens Negative Screen + Adeno Flu A Flu B Para 1 Para 2 Para 3 RSV Comparar Results Subject Results PPA 95% CI PPA 98.0% 73.4% 95.2% 83.1% 55.7% 45.4% 65.5% 91.3% NPA 95% CI NPA 98.5% 97.3% 95.0% 97.1% 97.4% 97.4% 96.6% 96.6% Specimens and culture isolates used in these studies came from nasopharyngeal (NP) aspirates, washes, swabs, bronchial alveolar lavages (BAL) and/or tracheal aspirates. Table 6 summarizes the participant age demographics according test results for a population of 326 fresh specimens, prospectively collected and evaluated for performance using the Comparar assay (see Study 1-DS Direct Specimen Method below). D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 18 of 30

19 Table 6. Age Distribution Adeno Flu A Flu B Para 1 Para 2 Para 3 RSV Negative Totals < 1m* m 2y y 12y y 18y y 21y > 21y Not reported * m = months, y = years Prospectively Collected Specimens Clinical Study Sites 1, 2, and 3 generated data for Direct Specimen (DS) Testing according the study design briefly summarized for each site. Clinical Study Sites 2 and 3 generated data for Cell Culture (CC) Testing according study design as summarized for each site. Study 1-DS - Direct Specimen Method The study consisted of a tal of 329 fresh specimens submitted February through May, 2006, the laborary for respirary virus testing. Slides were prepared from PBS-washed cells from the fresh specimens and fixed according the prescribed procol. The slides were sred at 70 C until testing was performed. The slides were brought ambient temperature and stained in accordance with the procedure in the Comparar product insert (same procedure for both Subject and Comparar devices). Three (3) specimens were found contain insufficient numbers of cells for interpretation of DS stain results, leaving 326 specimens for evaluation. The results of this testing are summarized in Table 7 below. Table 7. Study 1-DS Direct Specimen Results 326 specimens Negative Screen + Adeno Flu A Flu B Para 1 Para 2 Para 3 RSV Comparar Results Subject Results PPA 95% CI PPA 95.1% 79.3% 87.3% 79.3% 29.0% 29.0% 79.3% NPA 98.3% 98.7% 96.7% 95% CI NPA 95.4% 99.5% 94.2% 93.0% 92.2% > 99.9% 95.2% 95.3% 90.5% 99.3% 94.2% D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 19 of 30

20 With the exception of 4 specimens, the DS test results were concordant for both the screen and the identification of the individual viruses; the Comparar device identified 4 specimens as being negative while the Subject device identified one as Flu B and three as Para 3 infections. All but one of the Para 3 specimens were confirmed by culture; the one Para 3, although strongly positive by the Subject assay, could not be cultured confirm it as a Para 3. The culture method was not performed on the rest of the specimens from this site. Study 2-DS Direct Specimen Method The study consisted of 192 specimens submitted the laborary for respirary virus testing during December 2005 through February 2006, with residual specimen material sred at 70 C from a few days 2 months. The frozen specimens were thawed and processed between 13 February 17 February 2006 according the procedure in the Comparar product insert (same procedure for both Subject and Comparar devices). Slides were prepared from the specimens according instructions detailed in the Comparar device s product insert. These slides were stained with both the Comparar and Subject devices and interpreted according the Comparar device s product insert procedure (same procedure for both Subject and Comparar devices). All of the frozen/thawed specimens had sufficient intact cells for interpretation. The results of this testing are summarized in Table 8 below. Table 8. Study 2-DS Direct Specimen Results 192 specimens Negative Screen + Adeno Flu A Flu B Para 1 Para 2 Para 3 RSV Comparar Results Subject Results PPA 95% CI PPA 91.5% 29.0% 84.% 38.3% 16.8% 16.8% 80.5% NPA 95% CI NPA 96.8% 99.5% 92.6% 96.2% 96.8% >99.9% 96.8% 96.8% 96.8% 89.4% The DS test results were concordant for both the Screen and the ID reagents. Study 2-CC Cell Culture Method The same 192 specimens that were evaluated by DS testing were also processed according the Comparar device s product insert procedure for cell culture (same procedure for both Subject and Comparar devices). Briefly, 200 µl from the specimens were inoculated on each of 4 monolayers of R-Mix Too FreshCells contained in shell-vials which were centrifuged for 60 minutes at 700xg and incubated for 24 hours at 35 C 37 C. The shell-vials were processed according instructions detailed in the Comparar device s product insert. The results of this testing are summarized in Table 9 below. D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 20 of 30

21 Table 9. Study 2-CC Cell Culture Results 192 specimens Negative Screen + Adeno Flu A Flu B Para 1 Para 2 Para 3 RSV Comparar Results Subject Results PPA 95% CI PPA 91.5% 38.3% 84.8% 38.3% 16.8% 16.8% 77.3% NPA 95% CI NPA % 96.8% 96.2% 96.8% > 99.9% 96.8% 96.8% 96.8% 96.4% The CC test results were concordant for both the Screen and the ID of the specific viruses. Study 3-DS Direct Specimen Method The study consisted of 298 specimens originally received by a hospital laborary in the eastern US for respirary virus testing during January through March 2006, with residual specimen material sred at 70 C from 3 6 months. The frozen specimens were sent Quidel, where they were thawed and processed between 30 May and 1 June 2006, according the Comparar device s product insert. All specimens used in the studies were tested by both the DS and CC procedures as detailed in the Comparar device s product insert; however, a tal of 16 specimens were inadequate for interpretation of DS stain results (15 were found contain insufficient numbers of cells, and one other specimen exhibited non-specific staining with the Normal Mouse Gamma Globulin DFA Reagent), leaving 282 specimens for evaluation. The DS results for these specimens tested using the Comparar and Subject devices are summarized in Table 10 below. Table 10. Study 3-DS Direct Specimen Results 282 specimens Negative Screen + Adeno Flu A Flu B Para 1 Para 2 Para 3 RSV Comparar Results Subject Results PPA 95% CI PPA 96.2% 55.7% 92.7% 77.3% 29.0% 29.0% 65.5% 87.9% 96.2% NPA 95% CI NPA % 92.7% 95.6% > 99.9% 96.2% 96.2% 95.9% 91.3% D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 21 of 30

22 The DS test results were concordant for both the Screen and the ID reagents. There were ten (10) specimens identified with co-infections as follows: three (3) Flu A+Para 3, one (1) Flu B+Para 2, one (1) Flu B+Para 3, one (1) RSV+Para 1, three (3) RSV+Para 3 and one (1) Adeno+Para 3. Because of the ten (10) co-infections, the Negatives and Positives add up 292 ID results. Study 3-CC Cell Culture Method The same 298 specimens that were evaluated by DS testing were also processed for CC testing according the Comparar device s product insert for cell culture using R-Mix Too FreshCells in 48/24-fill cluster plates. The results of this testing are summarized in Table 11 below. Table 11. Study 3-CC Cell Culture Results 298 specimens Negative Screen + Adeno Flu A Flu B Para 1 Para 2 Para 3 RSV Comparar Results Subject Results PPA 95% CI PPA 96.6% 67.9% 93.5% 81.0% 51.1% 38.3% 65.5% 87.6% NPA 95% CI NPA 97.3% 96.3% 93.2% 96.0% >99.9% 96.4% 96.5% 96.3% 95.5% The CC test results were concordant for both the Screen and the ID reagents. There were sixteen (16) coinfections as follows: three (3) Flu A+Para 3, one (1) Flu A+Para 1, one (1) Flu A+Para 2, two (2) Flu A+RSV, one (1) Flu A+Adeno, one (1) Flu B+Para 2, one (1) Flu B+Para 3, one (1) Flu B+RSV, one (1) RSV+Para 1, two (2) RSV+Para 3, one (1) Adeno+Para 1 and one (1) Adeno+Para 3. Because of the sixteen (16) co-infections, the Negatives and Positives in the table add up 314 ID results. Non-prospective archival specimens Due relative low prevalence of parainfluenza infections in populations of respirary specimens, few specimens in the studies detailed above were reactive with the Parainfluenza DFA Reagents. In order better demonstrate performance characteristics of the Parainfluenza DFA Reagents, frozen original specimens previously determined contain parainfluenza (types 1, 2, or 3) during the 2006 respirary season were obtained from an additional laborary, and were tested in an internal reference laborary using the Subject and Comparar devices by Direct Specimen method (Study 3a-DS; see Table 12, below). The same specimens were tested by Cell Culture method (see Table 13). Original results reported by the laborary were unknown the study investigar. Although the study design has a selection bias, this study offers further analytical information on the assay's ability detect parainfluenza viruses. Study 3a-DS Direct Specimen Method The study consisted of 30 specimens originally received by a hospital laborary in Italy for respirary virus testing during the period from Ocber 2005 through April 2006, with residual specimen material sred at - 70 C from 2 6 months. The frozen specimens were sent Quidel, where they were thawed and processed D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 22 of 30

23 between June 7 and 8, 2006, according the prescribed procol. All specimens used in the studies were tested by both the DS and CC procedures as detailed in the Comparar device s product insert; however, a tal of four specimens were found contain insufficient numbers of cells for interpretation of DS stain results, leaving 26 specimens. The DS results for these specimens tested using the Comparar and Subject devices are summarized in Table 12 below. Table 12. Study 3a-DS Direct Specimen Method 26 specimens Negative Screen + Adeno Flu A Flu B Para 1 Para 2 Para 3 RSV Comparar Results Subject Results PPA 95% CI PPA 78.4% 16.8% 51.1% 70.0% NPA 88.9% 85.7% 95% CI NPA 54.3% > 99.9% 79.3% 79.3% 79.3% 78.4% 73.4% 46.7% 99.5% With the exception of one specimen, the DS test results were concordant for both the Screen and the ID of individual viruses; the Subject device identified one specimen as positive for Para 3 while the Comparar device was negative for this specimen. 79.3% Study 3a-CC Cell Culture Method The same 30 frozen specimens that were evaluated by DS testing were also processed for CC testing according the Comparar device s product insert for cell culture using R-Mix FreshCells in 48/24-fill cluster plates. One specimen was found be unsuitable for CC testing because it was xic the monolayer of cells. The results of this testing are summarized in Table 13 below. Table 13. Study 3a-CC Cell Culture Results 29 specimens Negative Screen + Adeno Flu A Flu B Para 1 Para 2 Para 3 RSV Comparar Results Subject Results PPA 95% CI PPA 81.8% 38.3% 51.1% 73.4% NPA 95% CI NPA 62.8% 81.8% 81.8% 81.8% 79.3% 77.3% 62.8% 81.8% D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 23 of 30

24 The CC test results were concordant for both the Screen and the ID reagents. Non-prospective archival clinical isolates To further demonstrate the proficiency of the Screening and Typing Reagents in the Subject Device, a study was conducted using a collection of banked clinical isolates known contain respirary viruses that had been frozen from the 2005/2006 respirary season. These specimens were selected because they were previously shown contain at least one of the seven virus analytes detected by the Subject Device. Study 3b-CC Cell Culture Method A tal of 81 clinical isolates from a frozen archival reposiry were processed according the Comparar device s product insert for cell culture using R-Mix FreshCells cultures in shell-vials. The results of this testing are summarized in Table 14 below. Table 14. Study 3b-CC Cell Culture Method 81 specimens Negative Screen + Adeno Flu A Flu B Para 1 Para 2 Para 3 RSV Comparar Results Subject Results PPA 95% CI PPA 94.6% 70.% 79.3% 78.4% 45.4% 16.8% 84.8% 51.1% NPA 95% CI NPA 97.3% 93.8% 93.1% 93.2% 94.3% 94.5% 92.2% 94.2% The CC test results were concordant for both the Screen Reagent and the specific virus ID Reagents. Because of the one co-infection, Para 1+ Para 3, the positive ID results added up 82. Cross-reactivity Testing D 3 Ultra DFA Respirary Virus Screening & ID Kit DFA Reagents were tested for cross-reactivity against a wide variety of cells and microorganisms. No cross-reactivity was observed for 64 virus strains (cultured and processed for staining) or for 18 host culture cell types. Nineteen (19) bacterial cultures were stained and examined for cross-reactivity, including Staphylococcus aureus, a protein-a-producing bacterium. Staining of S. aureus appeared as small points of fluorescence (see LIMITATIONS OF PROCEDURE) while all other bacterial cultures were negative. [See Table 15 for cross-reactivity study results. The table indicates which organisms were reactive with which DFA Reagent.] Stringent conditions for cross-reactivity testing were achieved by using high concentration of the DFA Reagents and high titers of microorganisms. The DFA Reagents (i.e., directly fluoresceinated monoclonal antibodies) were prepared at 1.5X the concentration that is provided in the kit. Each of the tested viruses was prepared as infected cell monolayers (250 infected cells inoculated in a shell-vial culture and incubated for hours, yield a infection), and processed and stained with the 1.5X DFA Reagents according the procedure detailed in this product insert. Bacterial strains were cultured, processed as suspensions, then D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 24 of 30

25 spotted on microscope slides (yielding > 150 bacteria per 400X microscope field), then stained with the 1.5X DFA Reagents according the procedure in this product insert. Cell cultures were stained as confluent monolayers. Table 15. Cross-Reactivity Study Results DFA Reagent (Results are Positive (+) or Negative ( ) for Reactivity Organisms Strain Adeno Flu A Flu B Para 1 Para 2 Para 3 RSV Type 1 + Type 3 + Type 5 + Type 6 + Type 7 + Adenovirus Type 10 + Type 13 + Type 14 + Type 18 + Type 31 + Type 40 + Type 41 + Mexico/4108/2009 (H1N1) from CDC* + California/07/2009 (H1N1) from CDC* + Aichi (H3N2) + Mal (H1N1) + Influenza A Hong Kong (H3N2) + Denver (H1N1) + Port Chalmers (H3N2) + Vicria (H3N2) + New Jersey (H SWN1) + WS (H1N1) + PR (H1N1) + Hong Kong + Maryland + Influenza B Mass + Taiwan + GL + Russia + Parainfluenza 1 C-35 + Parainfluenza 2 Greer + Parainfluenza 3 C Parainfluenza 4a M-25 Parainfluenza 4b CH19503 Long + RSV Wash D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 25 of 30

26 Rhinovirus Picornavirus Towne Cymegalovirus Davis AD169 Organisms Strain Adeno Flu A Flu B Para 1 Para 2 Para 3 RSV A1 Metapneumovirus A2 B3 B4 Coronavirus OC43 229E Herpes simplex 1F virus Type 1 MacIntyre Herpes simplex MS virus Type 2 Strain G Varicella-zoster Webster Ellen 9 Echovirus B1 B2 Coxsackievirus B3 B4 B5 B6 Mumps Rubeola Rhinovirus 209 Picornavirus BACTERIA Adeno Flu A Flu B Para Para Para RSV Acholeplasma laidlawii Bordetella bronchiseptica Bordetella pertussis Chlamydia pneumoniae Clostridium diphtheriae Haemophillus influenzae type A Klebsiella pneumoniae Listeria pneumophila Moraxella cartarrhalis Mycobacterium avium Mycobacterium intracellulare Mycoplasma hominis type 1 Mycoplasma orale Mycoplasma pneumoniae Mycoplasma salivarium Pseudomonas aeruginosa D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 26 of 30

27 Strepcoccus pneumoniae Strepcoccus pyogenes Ureaplasma urealyticum CELL CULTURES Adeno Flu A Flu B Para 1 Para 2 Para 3 RSV A549 BGMK HEp-2 LLC-MK2 MDCK MRC-5 MRHF Mv1Lu NCI-H292 pcmk prhmk prk RD RhMK II R-Mix R-Mix Too Vero WI-38 * Although this test has been shown detect the 2009 H1N1 influenza virus in two cultured isolates, the performance characteristics of this device with clinical specimens that are positive for the 2009 H1N1 influenza virus have not been established. The D 3 Ultra DFA Respirary Virus Screening and ID Kit can distinguish between influenza A and B viruses, but it cannot differentiate influenza subtypes. ASSISTANCE To place an order or for technical support, please contact a Quidel Representative at (in the U.S.) or (outside the U.S.), Monday through Friday, from 8:00 a.m. 5:00 p.m., Eastern Time. Orders may also be placed by fax at (740) For support contact cusmerservice@quidel.com or technicalsupport@quidel.com. For services outside the U.S.A., please contact your local distribur. Additional information about Quidel, our products, and our distriburs can be found on our website quidel.com. REFERENCES 1. FDA Guidance Document: In vitro Diagnostic Devices Detect Influenza A Viruses: Labeling and Regulary Path; Issued 4/10/ Englund, J.A., (2002). Antiviral therapy of influenza. Semin. Pediatr. Infect. Dis., 13(2): Patel, N., Hartwig, R., Kauffmann, L. and Evans, M. (2000). Rapid influenza A and B culture 20-hour detection using R-Mix: A new gold standard. Presented at The Sixteenth Annual Clinical Virology Symposium, April 30-May 3, Clearwater Beach, FL. 4. Rodriguez, W.J., Schwartz, R.H. and Thorne, M.M. (2002). Evaluation of diagnostic tests for influenza in a pediatric practice. Pediatr. Inf. Dis. J., 3: Gould, I.M. (2002). Antibiotic Policies and control of resistance. Curr. Opin. Infect. Dis., 15(4): Bischofberger, N., Webster, R.G. and Laver, G. (1999). Disarming Flu Viruses. Scientific American, January. D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 27 of 30

28 7. Foy, H.M. (1997). Adenoviruses. In: Evans, A., Kaslow, R., eds. Viral Infections in Humans: Epidemiology and Control. 4 th ed., New York, Plenum, Easn, A.J., Eglin, R.P. (1989). Epidemiology of Parainfluenza virus type 3 in England and Wales over a 10 year period. Epidemiol. Infect., 102: Fete, T.J., Noyes, B. (1996). Common (but not always considered) viral infections of the lower respirary tract. Pediatr. Ann., 25:10), Hall, C.B. (1981). Respirary Syncytial Virus. In: Feigin, R. D., Cherry, J.D., eds. Textbook of Pediatric Infectious Diseases, Phila., W.B. Saunders, Hall, C.B., Hall, W.J., Gala, C.L., MaGill, F.B., Leddy, J.P. (1984). Longterm prospective study in children after Respirary Syncytial Virus infection. J. Pediatr., 105: Falsey, Ann R. and Walsh, E.E. (2000). Respirary Syncytial Virus Infection in Adults. Clinical Microbiology Reviews 13(3): Biosafety in Microbiological and Biomedical Laboraries (BMBL), 5 th edition, 2007, CDC-NIH manual Biosafety Manual, 3 rd edition, World Health Organization [Manual may be available in additional languages; refer WHO web page Laborary Biosafety Guidelines, 3 rd edition, Published by authority of the Minister of Health, Population and Public Health Branch, Centre for Emergency Preparedness and Response [Guideline is available in French or English; refer web page [ 04/index.html] 16. Clinical and Laborary Standards Institute. Viral Culture; Approved Guidelines. CLSI document M41-A [ISBN ]. Clinical and Laborary Standards Institute, 940 West Valley Road, Suite 1400, Wayne, PA , USA Eisenberg, Henry D. (1992). Clinical Microbiology Procedures Handbook, published by American Society for Microbiology, Washingn DC, pg Clinical and Laborary Standards Institute. Viral Culture; Approved Guidelines. CLSI document M41-A [ISBN ]. Clinical and Laborary Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania , USA 2006, Section Clinical and Laborary Standards Institute. Viral Culture; Approved Guidelines. CLSI document M41-A [ISBN ]. Clinical and Laborary Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania , USA 2006, 20. Isenberg, Henry D., Clinical Microbiology Procedures Handbook, published by American Society for Microbiology, Washingn DC, Leland, Diane S. (1996). Clinical Virology, published by W.B. Saunders, Philadelphia, PA. D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 28 of 30

29 v2 D 3 Ultra DFA Respirary Virus Screening & ID Kit MDSS GmbH Schiffgraben Hannover, Germany Diagnostic Hybrids, Inc. a subsidiary of Quidel Corporation 2005 East State Street, Suite 100 Athens, OH USA quidel.com PI EN00 (10/18) D 3 Ultra DFA Respirary Virus Screening & ID Kit Page 29 of 30